The goal of this project is to understand how proteins are transported across the endoplasmic reticulum (ER) membrane in a posttranslational manner. This process must differ in fundamental mechanistic aspects from that of the better understood cotranslational mode. We have recently established a reconstituted system that reproduces the posttranslational pathway of protein transport with proteoliposomes containing a purified heptameric complex of ER proteins from S. cerevisiae. This Sec- complex contains a heterotrimeric Sec61p-complex, similar to that in mammals, but also all other membrane proteins found in genetic screens for translocation components. Efficient posttranslational transport also requires lumenal Kar2p (BiP) and ATP. We now propose to use this system to determine the molecular mechanism of posttranslational transport, to compare it with the cotranslational one and to determine how the two systems interact. Specifically, we will address the following questions: 1. Are distinct protein complexes involved in co- and posttranslational pathways? We will test the hypothesis that the Sec61p- complex serves as a core component that associates with the other Sec- proteins as well as with Kar2p in the posttranslational pathway, and functions together with the SRP-receptor in the cotranslational pathway. To this end, we will characterize and improve the reconstituted posttranslational system, and establish reconstituted systems that reproduce the cotranslational pathway in yeast. 2. What is the mechanism of posttranslational protein transport? Using the reconstituted systems and employing various other methods as well as mutants in the Sec- proteins, we will study how the signal sequence is recognized in the posttranslational translocation pathway, how the process is energetically driven, whether there exists a protein- conducting channel, and whether the channel is gated by Kar2p or other proteins. 3. What other factors are involved in translocation? We will analyze the function of a recently discovered, putative, second Sec61p- complex in yeast, identify cytosolic factors involved in the posttranslational translocation pathway, search for a homolog of the mammalian TRAM protein in yeast, and determine the primary structure and functions of the four subunits of the signal peptidase complex that have not yet been characterized. These studies will lead to a significant progress in our understanding of the first steps in the biosynthesis of secretory proteins. Although the project will be confined to the yeast S. cerevisiae, it is likely that the results will be of significance for the co- and posttranslational protein transport pathways in all organisms.